ABSTRACT Freshwater mussel conservation efforts by many federal and
state agencies have increased in recent years. This has led to a greater
number of stream surveys, in which mussel die-offs involving high
numbers of dead and moribund animals are being observed and reported
with greater frequency. Typically, die-offs have been incidentally
observed while research was being done for other purposes, therefore,
accurate mortality data have been difficult to obtain. Specifically,
seasonal die-offs were noted in localized areas of the Clinch and
Holston Rivers, Virginia, and to lesser degrees, in neighboring rivers
in this geographic region, including southeast Virginia. The observed
mussel species affected were primarily the slabside pearlymussel
(Lexingtonia dolabelloides) and to lesser extents, the pheasantshell
(Actinonaias pectorosa), rainbow mussel (Villosa iris), and the
endangered shiny pigtoe (Fusconaia cor). To determine if a bacterial
pathogen might be involved in these recurring mussel die-offs, this
study examined characteristics of the indigenous microbiota (bacteria)
from healthy mussels from sites on the Clinch and Holston Rivers where
die-offs were previously observed. These baseline data will allow for
recognition of bacterial pathogens in future mussel die-offs. Means for
total bacteria from soft tissues ranged from 1.77 x [10.sup.5] to 3.55 x
[10.sup.6] cfu/g, whereas, the range in means from fluids was 2.92 x
[10.sup.4] to 8.60 x [10.sup.5] cfu/mL. A diverse microbiota were
recovered, including species that are common in freshwater aquatic
environments. The most common bacterial groups recovered were motile
Aeromonas spp. and nonfermenting bacteria. Flavobacterium columnare, a
pathogen to cool- and warm-water fishes was recovered from one specimen,
a Villosa iris from the Clinch River.

Freshwater mussels native to the United States are one of the more
imperiled fauna (Lydeard et al. 2004, Williams et al. 1993). In light of
this, many federal and state agencies have implemented or increased
conservation efforts towards imperiled mussel species in recent years.
In conjunction with the greater numbers of stream surveys or similar
fieldwork being conducted, an increased frequency of apparently natural
mussel die-offs have been observed in rivers and streams across the
United States (Neves 1987). These events are characterized by high
numbers of dead and moribund mussels observed in their natural
environments, and typically are limited to a relatively localized
geographic area. Anecdotal observations indicate that the mortality
occurs within a time period ranging from about 2-4 days up to about two
weeks, which may be indicative of an infectious agent-induced disease.
This death rate generally differs from a rate that might be anticipated
from a noninfectious cause, such as critically low dissolved oxygen or
toxic substance exposure, in which an acute die-off might occur within a
much shorter timeframe, perhaps within one day. Because these natural
die-offs were typically noted incidentally and whereas related mussel
field research was being conducted, accurate mortality data have been
difficult to collect. Often, by the time an epizootic was noted,
explanatory evidence towards determining a cause was likely to have been
diluted and carried away with the flowing water. Furthermore, the gaped
valves of dead animals expose the soft tissues and this renders them
useless for any attempt to isolate a culturable pathogen.

Diagnosing the cause(s) of mussel die-offs among wild populations
is difficult because of the nature of observing mortality in localized
areas of large open waters and the relative lack of knowledge about
pathogens to freshwater mussels. This contrasts with the diseases
infecting some marine bivalves. Certain marine species are an important
aquaculture commodity and have a sustained commercial fishery to support
it. With captive rearing, diagnosticians have the opportunity to observe
behavioral changes in animals and possibly recognize diseases in their
early stages. The mortality can be observed and fresh specimens are
readily obtained for laboratory diagnostics. Subsequent descriptions of
causes for diseases and mortality may be made. Refuge propagation of
imperiled freshwater mussels is likely to continue increasing in future
years. Infectious agent-induced diseases could become problematic,
similar in the way they have with other organisms propagated at culture
facilities; for example, among cultured fishes (Noga 1996, Woo &
Bruno 1999, Wedemeyer 2001). Currently, about 15 federal and state
facilities (e.g., fish hatcheries) are maintaining and propagating a
suite of freshwater mussel species for use in restoration or
augmentation of wild populations. Effective disease preventative
measures are likely to become essential for the success of these
programs.

Along with the apparent death rate, additional observations from
die-offs lead to suspicions of pathogens as the causes of death. At
certain locations on the Clinch and Holston Rivers, Virginia, and to
lesser degrees in neighboring rivers, die-offs have been observed during
the same timeframes of different years, coinciding with seasonal
increases in water temperatures that likely favor optimal bacterial
pathogen growth and infectivity. Host specificity, or predilection for
certain mussel species is also thought to be a contributing factor to
disease. Mussel die-offs in the Middle Fork Holston and Clinch Rivers,
Virginia, have occurred in recent years during late spring and summer.
This encompasses spawning and glochidia release times for some mussel
species, including the endangered shiny pigtoe (Fusconaia cor), slabside
pearlymussel (Lexingtonia dolabelloides), a candidate species for
federal protection, the pheasantshell (Actinonaias pectorosa), and
rainbow mussel (Villosa iris). Large numbers of empty shells are common
in the affected stretches of the rivers. At this time, additional
specimens observed were apparently moribund as evidenced by delayed and
weakened hinge/valve closing in response to stimuli.

In an effort to delineate the possible role of a bacterial pathogen
in these die-offs, the approach taken in the current study has been to
describe the normative microbiota of healthy mussels from the affected
regions of these rivers. These baseline data, including total bacterial
loads in mussels and bacterial species profiles, will aid in the
recognition of suspected bacterial pathogens isolated from mussels
during subsequent die-offs. Describing the pathogens and diseases of
freshwater mussels is essential to developing disease prevention
strategies and procedures. This forms the basis for an effective health
management program designed to eliminate or greatly reduce the risk for
transmission of pathogens and spread of diseases among captive
populations at refuges, and to feral populations of mussels through the
stocking of refuge-reared individuals.

MATERIALS AND METHODS

Three species of mussels were collected by snorkeling from the
Holston and Clinch Rivers, Virginia: pheasantshell (Actinonaias
pectorosa), slabside pearlymussel (Lexingtonia dolabelloides), and
rainbow mussel (Villosa iris). These species and the collection sites
were selected for this study because of observed mussel die-offs at
these locations in recent years, particularly affecting populations of
L. dolabelloides, and to lesser degrees, the other two species. The A.
pectorosa and L. dolabelloides were collected from the Middle Fork
Holston River, river mile 10.1, Washington County, Virginia. The A.
pectorosa and V. iris from the Clinch River were collected at
Nash's Ford, river mile 279.8, Russell County, VA. Each location
was sampled in early summer (a die-off was not observed during this
year) and early fall within the same calendar year. After collection,
the mussels were kept alive and fresh during overnight shipment to the
National Fish Health Research Laboratory (Kearneysville, West Virginia)
by wrapping them in wet burlap and placing them in coolers at the
collection site. The mussels were sampled for bacteriological analyses
immediately upon arrival at the laboratory. A total of 80 mussels were
examined for the presence of indigenous bacterial.

Primary isolation of bacteria from each mussel was completed using
the methods described in Starliper et al. (1998) and Starliper (2001).
External surfaces of the valves were cleaned and disinfected by gentle
scrubbing with a brush and a solution of 200 mg/L sodium hypochlorite.
The following morphometric data were recorded for each specimen: length,
width, depth, total weight, and the area inside the valves (determined
by measuring the volume of water necessary to fill the valves where 1 mL
= 1 cc area). Aseptic techniques were used to the extent possible to
collect the fluids and soft tissues used for bacterial sampling. The
valves were pried 12-15 mm apart so the adductor muscles could be cut.
The fluid (liquid inside the valves) was poured into a sterile Petri
dish, and measured volumetrically. The soft tissues were excised and
treated as one sample (per animal). The outer surfaces of the soft
tissues were disinfected by submersing them in 1L of 200 mg/L sodium
hypochlorite and gently keeping them in motion using a sterile pipette
for 30 s. They were immediately rinsed for 5-10 s in sterile pep-ye
(0.1% peptone, 0.05% yeast extract; Difco, Becton, Dickinson and
Company, Sparks, MD) and placed in a sterile sampling bag (Fisher
Scientific, www.fishersci.com). The tissues were then homogenized in an
equal amount (w/v; 1:2 dilution) of sterile pep-ye for two min using a
Model 80 Laboratory Blender (Seward Medical, London, UK). Three serial
10-fold dilutions in pep-ye were made from each fluid sample and from
each tissue homogenate. Standard volumes (three 0.025 mL drops) of the
fluid samples, tissue homogenates, and all dilutions were used to
drop-inoculate the surface of two bacteriological media poured in Petri
dishes, brain heart infusion agar (BHIA; Difco) and R2A agar (Difco).
The plates were incubated until the growing bacterial colonies were
large enough to comfortably enumerate and select for transfers.
Incubation was for 24-72 h; BHIA plates were incubated at 20[degrees]C
to 22[degrees]C, and R2A plates were incubated at 16[degrees]C. The
total numbers of colony-forming units cultured from each tissue (cfu/ g)
sample and fluid (cfu/mL) were calculated by averaging the numbers of
colonies enumerated (from three 0.025 mL standard volumes) from the
lowest sample dilution that yielded single, isolated colonies, which
were easily and accurately scored. The mean was multiplied by the
dilution factor to convert to the number in standard units (per mL or
per g). Bacterial colonies from BHIA and R2A media representing all
colony morphologies were selected and transferred to fresh media for
growth. These resulting cultures were checked for purity by
streak-plating and each was identified using standard biochemical and
characterization methods (Griffin 1992, Holt et al. 1994, Janda &
Abbott 1998, Koneman et al. 1992, MacFaddin 2000, Murray et al. 1999)
and the API identification system (bio-Merieux, Inc., Hazelwood, MO).
Significant differences ([alpha] = 0.05) among the morphometric and
bacterial cfu data were identified using analysis of variance (ANOVA)
and the z-statistic. The morphometric data were evaluated for
differences associated with collection sites (Holston River versus
Clinch River) and collection dates (summer versus fall).

RESULTS

The means and ranges for the morphometric data from the three
species of mussels, A. pectorosa, L. dolabelloides, and V. iris are
presented in Table 1. The largest of the three species was A. pectorosa;
the mean total weights for 10 specimens ranged between 208.4 g for the
Clinch River summer collection and 294.5 g for the fall group from
Holston River. The smallest A. pectorosa of the study weighed 142.6 g,
whereas the heaviest was 447.5 g. From the summer and fall collection
dates, the means for the total weights of A. pectorosa groups from the
Holston River were greater than those for groups from the Clinch River.
However, the means for percent of the mussels' total weight
comprised of soft tissues or fluids from the Clinch River groups were
larger than cohort groups from the Holston River. Regardless of the
river of origin and collection date, the combined weight of soft tissues
and fluid comprised about one fifth of the mussels' total weight.
The mean total weights of A. pectorosa collected in the fall, from the
Holston and Clinch Rivers were greater than those from the summer
collections. This contrasted with the other two mussel species in which
the means for total weights were greater in summer. The total weight of
L. dolabelloides, comprised of soft tissues and fluids, was greater in
fall (23.0%) compared with summer (15.8%). This contrasted with V. iris
where the percentage in summer (28.9%) was greater than that for fall
(27.2%). The smallest of the three mussel species in total weight was V.
iris; however, the percentages of their total weight comprised of soft
tissues and fluids, combined, were greater than those for A. pectorosa
and L. dolabelloides.

Statistical comparisons of morphometric data by river and
collection date are given in Table 2. Of six possible pairings of river
and date for comparison, only one differed by total weight; A. pectorosa
collected in fall from the Holston River were larger (P = 0.006) than
those collected from the Clinch River. Of the 42 individual comparisons
listed in Table 2, significant differences were noted in 12 instances.
Only one of these was a comparison that did not involve A. pectorosa;
the percent of the total weight of L. dolabelloides comprised of fluid
was greater (P < 0.001) in fall (12.3%; Table 1) than in summer
(4.2%). Eight of the 12 significant pairings were attributed to seasonal
(summer versus fall) comparisons. The summer versus fall collection
dates for A. pectorosa from the Clinch River accounted for the largest
number (4) of significant differences, with differences in length (P =
0.030), depth (P = 0.025), tissue percent weight (P < 0.001), and
fluid percent weight (P = 0.002). Furthermore, the means for length,
depth, and tissue percent weight were greater in fall, whereas the mean
for fluid percent weight was greater in summer, 10.2% relative to 7.5 %
(Table 1). Collection dates for A. pectorosa from the Holston River
accounted for three significant differences; the means of each were
greater from the fall collection, length (P = 0.001), width (P = 0.007)
and depth (P = 0.020) than from the spring collection. There were no
significant differences in morphometric data for area, and for V. iris
from summer versus fall collections from the Clinch River.

The means and ranges for quantities of total bacteria isolated from
the mussels' fluids and tissues are given in Table 3. There were no
significant differences between the two bacteriological media (BHIA and
R2A) for quantitative recovery of total bacteria isolated from fluids (P
= 0.682) or tissue homogenates (P = 0.556). Each of the means for total
bacteria from the tissue homogenates was greater than the means from
paired fluids; seven of the eight tissue homogenate means were
significantly greater (P values ranged from <0.001-0.015), and
although the mean from A. pectorosa tissues from the (Clinch River) fall
sampling was greater than in summer, it was not significantly different
(P = 0.164). The highest total bacterial recovery was from the tissues
and fluids from A. pectorosa and V. iris during the Clinch River summer
collections with a minimum of 2.41 X [10.sup.6] cfu/g from tissues and
at least 1.63 X [10.sup.5] cfu/mL from fluids. The largest difference
between tissue and fluid cfu was from V. iris in summer, the differences
were 2.25 x [10.sup.6] cfu with BHIA, and 3.32 x [10.sup.6] cfu using
R2A.

The means for bacteria isolated from tissues ranged from 1.77 x
[10.sup.5] cfu/g to 3.55 X [10.sup.6] cfu/g. The range from fluids was
2.92 x [10.sup.4] cfu/mL to 8.60 x [10.sup.5] cfu/mL. The highest total
bacterial recovery from a tissue homogenate from a specimen, a V. iris,
was 1.52 x [10.sup.7] cfu/g, and the highest from a fluid sample was
nearly 10-fold less at 4.80 x [10.sup.6] cfu/mL from an A. pectorosa.
Comparisons of the means for bacteria recovered from mussels to
determine significant differences because of river of origin or
collection date are presented in Table 4. During summer collections of
A. pectorosa from the two rivers, bacterial recovery from tissues (P =
0.007) and fluids (P < 0.001) was greater from the Clinch River
specimens. Total bacterial loads from A. pectorosa collected in summer
from the Clinch River were also significantly higher (tissues: P =
0.002; fluids: P < 0.001) than the means in bacteria from A.
pectorosa collected from the same river in fall. From the Holston River,
A. pectorosa tissues from the fall collection yielded significantly
higher cfu/g (P = 0.019) compared with summer, and also produced a
greater mean for total bacteria from tissues relative to cohorts from
the Clinch River (P = 0.003). Tissue homogenates from L. dolabelloides
resulted in significantly higher cfu/g (P = 0.002) in the fall, with no
difference (P = 0.829) in bacterial cfu/mL from the paired fluids. There
were no significant differences in the total bacteria isolated from
tissues (P = 0.949) or fluids (P = 0.180) from V. iris between the two
collection dates.

A diverse microbiota was recovered from the mussels from the
Holston and Clinch Rivers (Table 5). There were at least 30 different
bacteria present in mussels throughout this study; 13 of which were
recovered from both rivers. This broad range in bacteria was noted in
summer and fall collections and from both tissues and fluids (data not
shown). The predominant bacteria in animals from both rivers were motile
Aeromonas spp. and various glucose-nonfermenting bacteria, including
Acinetobacter species, Brevundimonas vesicularis, Chryseobacterium
indologenes, Pseudomonas fluorescens, and Sphingomonas paucimobilis. A
variety of enteric bacteria were also identified, including Citrobacter
koseri, Enterobacter intermedius, Hafnia alvei, Proteus vulgaris,
Providencia rettgeri, and Serratia spp. Flavobaeterium columnare, a
pathogen to many warm- and cool-water fishes (Noga 1996, Wedemeyer
2001), was isolated from a single V. iris from the Clinch River during
the summer collection. This Gram-negative bacterium was isolated on R2A
medium from the fluid sample, bur not from the paired soft tissues
homogenate. Furthermore, this specimen was one of the smaller mussels
(total weight: 4.75 g) collected during the study. On the R2A primary
isolation plate, 4.00 x [10.sup.3] F. columnare per mL was cultured, and
0.3 mL of fluid was recovered, therefore, a total of 1.20 x [10.sup.3]
viable cfu F. columnare was calculated to be present in that specimen.

DISCUSSION

Historic numbers of many species and populations of freshwater
mussels native to the United States have declined, and continue to
decline, because of causes that either directly affect the mussels or
impact hosts for transformation of glochidia (Lydeard et al. 2004;
Williams et al. 1993). Mussel conservation efforts to salvage or sustain
many of the imperiled species are ongoing. One strategy involves
collecting at-risk individuals from impacted rivers and relocating them
to refugia for propagation. Success at propagation will depend on the
development and implementation of techniques that ensure good mussel
husbandry, including proper diet and feeding, adequate water quality and
flow, proper substrate, and identification of hosts for transformation.
The objective for propagation is to provide healthy individuals for
future strategic stockings into impacted rivers for augmentation or
restoration of affected species or populations.

Federal and state fishery agencies rely on periodic health
inspections of cultured fishes as an element of disease prevention
programs. For example, the United States Fish and Wildlife Service has
regional fish health units that specialize in proper husbandry and
recognition of common and emerging fish pathogens and diseases. Routine
health inspections are essential for controlling pathogen introductions
to production facilities and to naive, feral populations through
stockings into streams. These Fish Health Units also conduct extensive
health evaluations on wild fish populations (National Wild Fish Health
Survey; www.fws.gov/wildfishsurvey) to determine the prevalences of
select pathogens, and the host and geographic ranges of diseases. This
important information allows resource managers to make well-informed
decisions about relocating or stocking fishes between rearing
facilities, and to feral streams. A program similar to this for mussels
could provide important information to mussel resource managers.

Because mussels continue to be collected from open waters and
introduced to refuges, this presents a continuing risk of pathogens to
resident mussels at refuges. The consequences for a pathogen
introduction scenario to hatchery-reared fish are well documented (Noga
1996, Wedemeyer 2001), and a similar process could occur for captive
mussels. Another complicating factor is many of the host fishes required
for glochidia transformation are not commonly propagated. Therefore,
these fish must also be wild-caught and placed at refuges, which
heightens the risk of introducing pathogens, to resident fish and
perhaps, to mussels.

The profile of bacteria within mussels apparently changes quite
rapidly, which can be useful to prevent pathogen introductions. Nichols
et al. (2001) identified if endosymbiotic microbes (i.e., bacteria) were
present in mussels; they were not present. They described bacteria in
mussels to be transient, which can imply that the microbiota is subject
to relatively quick change in response to a change in environment.
Furthermore, they showed that the bacteria in mussels varied by season
and habitat, and not by mussel species. If mussels collected from rivers
for refugia are first quarantined (e.g., 30 d; Chaffee 1997, Gatenby et
al. 1998) to guard against the introduction of zebra mussels, this
quarantine could also offer the opportunity for mussels to depurate
pathogens. Previous studies with three-ridge (Amblema plieata) and
ebonyshell (Fuseonaia ebena) have also shown that the microbiota of
mussels responds rapidly to changes in their water supply (Starliper et
al. 1998). Using a model system and the fish pathogenic bacterium
Aeromonas salmonicida, it was shown that the percentage of A. plicata
that were A. salmonicida-positive was reduced from 100% to 0% within 30
d in a flow-through water system (Starliper 2001). This demonstrated
that quarantine for zebra mussels might also be effective in greatly
minimizing the risk of transmitting pathogens into the captive
population. However, extended periods of quarantine can result in a
reduced condition factor (Patterson et al. 1997, 1999), which might
serve to predispose mussels to diseases and minimize the effect of the
quarantine.

It is important to develop knowledge on the characteristics of the
indigenous microbiota in healthy mussels, because this will serve as the
expected, or baseline, when diseased animals are examined. Whereas the
microbiota from the mussels of a certain area may typify that particular
geographic locale, some generalized observations can be made for
nondiseased mussels. There is typically one-half to 10-fold greater
total bacterial load (cfu) present within tissues than is isolated from
the paired fluid samples. However, the microbiota present in fluids
reflects that which is isolated from the soft tissues. As would be
expected from other healthy aquatic animals such as fishes, the
microbiota in mussels is also quite diverse, usually at least several
bacterial species may be readily isolated from healthy mussels. A wide
range in bacterial species was isolated in the present study, and has
been noted in other mussel species, including several species from the
Ohio River (Starliper et al. 1998), and Elliptio complanata (Chittick et
al. 2001). An expected diverse microbiota is likely a useful criterion
to keep in mind when examining moribund or freshly dead mussels from a
die-off having a suspected bacterial etiology. In an epizootic offish,
for example, recovery of the bacterial pathogen causing the disease can
be expected from many of the individuals examined, and the pathogen cfu
recovery will be high relative to that from a normative microbiota.
Primary bacterial growth on bacteriological media will often show pure
or nearly pure cultures from diseased tissues, which contrasts with the
variety in bacterial species isolated from healthy individuals. High cfu
recovery of nearly pure cultures on the appropriate medium will be
indicative of a pathogen.

The extensive list of bacteria from mussels in the present study
(Table 5) is generally representative of common aquatic environmental
bacteria from freshwater, and some or most would likely be cultured from
the fluids and tissues of mussels from many different waters.
Acinetobacter spp., motile Aeromonas spp., Pseudomonas spp., Moraxella
sp., and enteric bacteria are noteworthy examples, because these genera
are also isolated from marine bivalves. Iida et al. (2000) isolated an
average of 1.00 x [10.sup.5] cfu total bacteria per gram from the
digestive tract tissues of Pacific oysters Crassostrea gigas. Some of
the same genera listed in Table 5 were isolated as part of the
microbiota from other marine bivalves including Mytilus
galloprovincialis, Perna viridis, and Seapharca cornea (Kueh & Chan
1985, Salati et al. 1999). Also, these bacteria are occasionally
isolated from the external (mucus) and internal tissues of apparently
healthy fishes, and some, in particular motile Aeromonas spp., P.
fluoreseens, S. liquefaciens, and S. putrefaciens, are recognized as
opportunistic or secondary pathogens to some species of cultured fishes,
including salmonid and catfish species. At present, none of these
bacteria are known to be the cause of any diseases to freshwater
mussels. In the present study, some of the bacteria were isolated from
the Holston and Clinch Rivers (Table 5), whereas others (e.g.,
Acinetobacter lwoffii in the Holston or Hafnia alvei in the Clinch) were
isolated from only one site. The microbiota can also be anticipated to
vary between origins and collection dates. For example, although A.
lwoffii was not isolated from the Clinch River during this study, its
presence in the Clinch River in future isolations would not necessarily
identify it as a pathogen unless other criteria were met. Heavy growth
of pure, or nearly pure cultures on primary bacterial isolation plates
from a significant number of moribund or fresh-dead individuals is one
characteristic that may lead to suspicion of the bacterium as a
pathogen.

The isolation of F. columnare from V. iris was the second such
reported isolation of this bacterium from a riverine mussel. Previously,
F. columnare was isolated from tissues of a three-ridge mussel Amblema
plicata from the Ohio River; however, it was not recovered from cohort
A. plicata after one day or more of depuration in a pathogen-free water
supply (Starliper et al 1998). There was a total of 1.20 x [10.sup.3]
cfu of F. columnare present in the positive V. iris in the current
study. This negligible quantity of viable cells would likely have been
quickly depurated if this mussel had been subjected to similar
quarantine parameters applied to the A. plicata in the previous study
(Starliper et al. 1998).

Any use of trade, product, or firm names is for descriptive
purposes only and does not imply endorsement by the United States
Government.

ACKNOWLEDGMENTS

This research was funded through the Quick Response program of the
United States Geological Survey and the United States Fish and Wildlife
Service, Department of the Interior. Janet Clayton and Craig Stihler of
the West Virginia Division of Natural Resources provided guidance on
mussel collection and importation permits. The authors thank Dr.
Christine Densmore and Dr. Barnaby Watten for their insightful reviews.